Method of evaluating green malt qualities by electron spin resonance spectrometry and method of evaluating malt qualities

ABSTRACT

A method is provided in which a correlation is made between a parameter indicating modification of a sampled malt in a germination process in which barley is germinated, and an ESR signal intensity of the sampled malt. The parameter is determined, in advance, by general analytical methods including chemical analytical methods. The ESR signal intensity is determined by measuring a peak height of spectrum of the sampled malt at a g value at which an unpaired electron derived from a carbon radical is resonant. The ESR signal intensity is a ratio of the measured peak height of spectrum to a peak height of spectrum of a reference, per a unit weight of the sampled malt. The modification state of the sampled malt can be evaluated based on the ESR signal intensity determined by electron spin resonance spectrometry. Kohlbach index, Hartong index at 45° C., diastatic power, β-glucan content, viscosity, and friability may be used as the parameter.

TECHNICAL FIELD

The present invention generally relates to a quality evaluation methodof malt to be used in the production of malt alcoholic beverages such asbeer, and, more particularly, to a quality evaluation method of maltusing Electron Spin Resonance (ESR) analysis.

BACKGROUND ART

Beer is mainly made of barley malt that serves as the source of starchand enzyme. The production process of beer is classified into a maltingprocess, a brewing process, and a packaging process. In the maltingprocess, malt is steeped into water for germination. During germination,degradative enzymes are activated. The germinating barley is calledgreen malt. After germinating to an expected extent of modification,green malt is dried (kilned). In the next brewing process, water isadded to ground malt and is heated for saccharification, and then, isfiltered. The filtered liquid is boiled after adding hop, andsaccharified liquid (wort) is obtained by separating hop dreg. Aftercooling, the wort is fermented by adding yeast, and is stored at a coldtemperature. After carbon dioxide generated during the storage isdissolved into the fermented liquid (beer), and flavor is matured, thebeer is filtered. In the packaging process, the beer is antisepticallyfiltered and is contained in barrels and/or bottles for distribution.

The quality of beer heavily depends on the quality control of themalting process, that is, the quality of produced malt. In general, agerminating process takes about four to six days. During germination,enzymes are synthesized, and starch and protein are partially degraded.The grade of malt degradation is referred to as “modification”.

In the production process of beer, it is important to control the maltquality. Kohlbach Index, Hartong index at 45° C., diastatic power,viscosity, and β-glucan content are indexes that indicate the quality ofmalt. These parameters are controlled in the malting process to maintainconstant malt quality.

The parameters for indicating malt modification are described below.

Kohlbach Index: the ratio of the nitrogen amount of congress wortprepared for analysis and the nitrogen amount of the whole malt. Itindicates the extent of protein degradation in the malt. The higher theKohlbach Index is, the more the malt is degraded. (modified).

Hartong index at 45° C.: the ratio of the extract of 45° C., 1-hourmashing of fine ground malt and the extract of the congress wort. Theamount of extract mainly depends on the amount of starch and sugar. Inmalt, the degradation of protein components existing among starchparticles affects the amount of extract. Accordingly, Hartong index at45° C. indicates the degradation grade of both starch and protein. Thehigher the Hartong index at 45° C. is, the more the malt is modified.

Diastatic power: To determine the starch degradative enzyme power ofmalt, the amount of reduced sugar generated by affectingwater-extraction liquid of malt to soluble starch is measured usingiodometry. The starch degradative enzymes are synthesized as the barleygerminates. In a brewing process, these enzymes are required fordegradation of starch in malt and in adjuncts (for example corn starch).Malt with low diastatic power may cause a problem in a beer brewingprocess.

β-glucan content: β-glucan is the degradative product of cell wall ofthe malt. If the cell wall is not degraded enough, the lautering of mashbecomes not smooth. Beer with high β-glucan content may cause haze afterfreezing.

The β-glucan content is measured as follows: after ground malt is heatedin ethanol to inactivate enzyme, β-glucan in the malt is degraded toglucose by processing with lichenaze and β-glucosidase; and the amountof generated glucose is determined using glucoseoxidase/paroxidasemethod.

Viscosity: the viscosity of congress wort prepared for the analysis.Polysaccharides such as starch, dextrin, β-glucan, and pentosan mainlyaffect the viscosity of wort. The viscosity indicates the degree ofdegradation of stored starch and cell wall. The lower is the viscosity,the more the malt is moderated. The viscosity is measured at 20.00° C.with Ubbelohde type viscometer.

Friability: Friability is measured by a dedicated friability meter.Specifically, the friability is measured as follows. After being groundwith a roller, malt samples are separated into one that passes through aspecific slit and one that does not pass through the specific slit andremains. The separated malt samples are measured. The friability of themalt samples is defined as the ratio between the separated malt samples.Malt of which starch and cell wall are not well degraded is crystallineand consequently hard. Friability of such malt is low. Well degradedmalt is mealy and easy to grind. The friability of such malt is high.

Conventionally, the above parameters for analyzing the modification ofmalt are determined with general analytical methods such as chemicalanalysis. However, the general analytical methods have problems suchthat the preparation and measurement of samples require considerabletime. A rapid and simple analytical technique for malt qualityevaluation is needed.

There are many analytical techniques for analyzing material, such aschemical analysis, optical analysis, and physical analysis (using X-ray,for example). Recently, electron spin resonance (ESR) analysis isdrawing attention as an analytical technique to obtain molecular levelinformation of material. The application of ESR analysis is beingintensively studied.

The principle of ESR is exactly the same as that of nuclear magneticresonance (NMR). ESR is a kind of magnetic resonance spectrum. Whereasthe NMR measures the resonant absorption of nuclear spins, the ESRmeasures the resonant absorption of electron spins. The ESR mostdirectly reveals information about the molecular structures and electronstates of materials having unpaired electrons such as a radical and atransition metal complex (trivalent iron ion and bivalent copper ion,for example). As of now, the ESR is the most reliable analyticaltechnique for detecting radicals.

ESR spectra show the following: g value indicating the position ofresonance of the unpaired electrons, resonance intensity indicating thenumber of unpaired electrons, absorption width related to relaxationtime, and hyper fine structure caused by the coupling between theunpaired electrons and atoms (¹H and ¹⁴N, for example) having nuclearspin near the unpaired electrons. Since each of radical species exhibitsan intrinsic position of absorption, one can identify the radicalspecies based on the g value. Additionally, one can consider reactiontime and reaction mechanism based on the change over time of absorption(intensity).

The inventors intensely studied the application of ESR to the evaluationof state (quality) of malt in beer brewing. As a result of the study,the inventors discovered that, if an absorption intensity (signalintensity) is observed, the radical species is roughly identifiablebased on the g value, and that there is a correlation between theabsorption intensity (signal intensity) and the state (quality) of greenmalt and malt. The present invention, which is made based on the abovediscovery, provides a more rapid and simpler method of evaluating thequality of malt, than that of conventional analytical techniques.

SUMMARY OF THE INVENTION

The above problems can be solved by means of the present inventiondescribed below.

According to an aspect of the present invention, a quality evaluationmethod of green malt sampled in a germinating process of barley byelectron spin resonance (ESR) spectrometry, includes the steps of:measuring a peak height of spectrum at a g value at which an unpairedelectron derived from a carbon radical is resonant; determining an ESRsignal intensity that is a ratio of the measured peak height of spectrumto a peak height of spectrum of a reference, per a unit weight of thesampled green malt; and evaluating a germination state of said sampledgreen malt by comparing the determined ESR signal intensity with apredetermined reference level.

The present invention makes it possible to determine the ESR signalintensity of green malt samples by using an electron spin resonancemethod, and to evaluate the quality of green malt samples based on thedetermined ESR signal intensity.

According to a second aspect of the present invention, aquality-evaluation method of malt sampled in a germinating process ofbarley by electron spin resonance (ESR) spectrometry, includes the stepsof: measuring a peak height of spectrum at a g value at which anunpaired electron of a carbon radical is resonant; determining an ESRsignal intensity that is a ratio of the measured peak height of spectrumto a peak height of spectrum of a reference, per a unit weight of thesampled malt; and evaluating a modification of the sampled malt bycomparing the determined ESR signal intensity with a predeterminedreference level.

The present invention makes it possible to obtain the ESR signalintensity of malt samples by using an electron spin resonance method,and to evaluate the quality of the malt samples based on the ESR signalintensity.

According to a third aspect of the present invention, an evaluationmethod of modification of malt sampled in a germination process ofbarley, includes the steps of: determining, in advance, a correlationbetween a parameter measured by general analytical methods includingchemical analytical methods, said parameter indicating modification ofthe sampled malt, and an ESR signal intensity of the sampled malt, theESR signal intensity being determined by an electron spin resonancespectrometry whereby a peak height of spectrum of said sampled malt ismeasured at a g value at which an unpaired electron of a carbon radicalis resonant, and the ESR signal intensity is a ratio of the measuredpeak height of spectrum to a peak height of spectrum of a reference, pera unit weight of the sampled malt, and evaluating said modification ofthe sampled malt based on said parameters determined using thecorresponding ESR signal intensity determined by said electron spinresonance spectrometry.

The present invention makes it possible to determine the ESR signalintensity of malt samples by using an electron spin resonance method,and to estimate parameters indicating the modification of malt based onthe determined ESR signal intensity. Accordingly, the quality of maltcan be evaluated.

At least one of a Kohlbach index, Hartong index at 45° C., diastaticpower, β-glucan content, viscosity, and friability is used as theparameter for indicating the modification of malt.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing an ESR spectrum of green malt;

FIGS. 2A and 2B are charts showing the effect of steep-out moisture andgermination time on the ESR signal intensity of green malt;

FIG. 3 is a chart showing the effect of germination time on the ESRsignal intensity of malt; and

FIGS. 4A through 4F are charts showing the correlation between variousparameters and the ESR signal intensity of malt.

BEST MODE FOR CARRYING OUT THE INVENTION

Embodiments of the present invention are described in more detail below.The outline of the ESR analyses performed by the inventors is describedfirst.

(1) Barley, Malt Sample

If much moisture is contained in the sample, the moisture absorbs theelectron spin resonance. When solid samples containing much moisture aremeasured with an ordinary ESR analysis method, the moisture needs to beremoved. Accordingly, green malt was used after being lyophilized, as asample for the analysis of germinating barleys (green malt). However,since malt contains low enough moisture, the malt was used as a samplewithout being lyophilized.

Japanese “Amagi Nijo” barleys and Canadian “Kendall” barleys were usedas the samples. They were malted with a 90 kg-scaled pilot maltingplant. The barleys were steeped in water at 14° C. and were germinatedat 14° C. Malt with different germination time was prepared using an 8kg-scaled kilning apparatus.

(2) ESR Analysis

The ESR analysis was carried out at the following measuring conditions.

-   -   Temperature of sample: room temperature    -   Sweep time: 60 seconds    -   Modulation width: 0.1 mT    -   Microwave power: 10.63 mW    -   Sweeps/measurement: 1 time    -   Internal standard: Mn²⁺

Five whole grains of green malt and malt (0.4 g), or about 0.2 g ofground samples of them were put in an ESR sample tube (cylindricaltube), and their electron spin resonance absorption was measured with anelectron spin resonance spectrometer. After the measurement of g valuebased on measured spectra, the signal intensity was calculated as therelative ratio of the peak height of the sample to the peak height ofMn²⁺ used as the internal standard. The ESR signal intensity is definedas the relative height per a unit weight of sample (/g). One sample wasmeasured 5 times and obtained data were averaged.

(3) ESR Preparatory Analysis of Green Malt and Malt

FIG. 1 is a graph showing an ESR spectrum of ground green malt.

As is shown in FIG. 1, the spectrum shows a singlet peak at the positionof g=2.0049. The g value g=2.0049 means that this spectrum is caused bycarbon radicals. It is well known that, in food samples, which containlipids and proteins (amino acid), for example, free radicals generatedby the oxidation of lipids take hydrogen from the proteins and generatestable organic radicals derived from proteins, and that they produce asinglet peak around g=2.00. Accordingly, the inventors speculate thatthis peak is also caused by stable organic radicals derived fromproteins in the green malt. In addition, ground malt samples and wholegrain samples of malt and green malt (without grind) showed similarspectra pattern.

(4) Change in ESR Signal Intensity of Green Malt During Germination

Another experiment was performed to determine change in the ESR spectrum(signal intensity) of green malt during malting (germination).

FIGS. 2A and 2B show the results of ESR measurement of the lyophilizedgreen malt sampled once a day after germination and lyophilized. FIG. 2Ashows the result of whole grains of green malt malted with two differentsteep-out moisture levels of 37% and 43%, and FIG. 2B shows the resultof ground green malt malted with steep-out moisture levels of 37% and43%.

These experimental results show that, as germination time passes, theESR signal intensity increases. The results also show that the higher isthe steep-out moisture level, the higher is the ESR signal intensity.Accordingly, it is found that the germination state of barley can beevaluated based on the ESR signal intensity.

From a comparison between the whole grain samples and ground samples,the ESR signal intensity of green malt increased with grind. Thisincrease in ESR signal intensity could be derived from the increase inradicals by oxidation during grinding.

Based on the above experimental results, the inventors speculate thatthe amount of radicals is increased during germination because thegermination activates the respiration of barley and consequently, theoxidation of ingredients contained in the barley, and accelerates thegeneration of stable organic radicals derived from proteins. Only thesignal intensity changed with the germination time in the spectra. Nonew peak is observed in the spectra. The increase in stable organicradicals derived from proteins during germination is an interestingdiscovery in view of various changes of ingredients including thesynthesis of enzyme in germinating barley. This discovery is expected tobring a new angle into the study of botanical germination.

(5) Relationship Between ESR Signal Intensity and Malt Parameters

Other green malts germinated for one through six days were kilned intomalts and ground. The ESR signal intensities of the ground malts werecompared.

FIG. 3 shows the result of the above experiment. As is shown in FIG. 3,the ESR signal intensity increased with germination time. Based on thisresult, the inventors confirmed that the increase in the ESR signalintensity of green malt with germination time is retained even afterkilning.

The correlation between several typical parameters indicating maltmodification and the ESR signal intensity was studied. FIGS. 4A through4F show the relationship between the parameters indicating maltmodification and the ESR signal intensity of malt samples used for themeasurement shown in FIG. 3. FIGS. 4A through 4F show the relationshipof malt quality parameters, that is, Hartong index at 45° C. (FIG. 4A),viscosity (FIG. 4B), β-glucan content (FIG. 4C), friability (FIG. 4D),diastatic power (FIG. 4E), and Kohlbach index (FIG. 4F), respectively,with the ESR signal intensity.

FIG. 4A shows that Hartong index at 45° C. increases with signalintensity less than 9.5, and remains constant with signal intensity of9.5 or more.

FIG. 4B shows that viscosity is constant with signal intensity of 12 ormore.

FIG. 4C shows β-glucan content does not remain constant over whole rangeof signal intensity.

FIG. 4D shows that friability remains constant with signal intensity of9 or more.

FIG. 4E shows that the diastatic power remains constant with signalintensity of 9 or more.

FIG. 4F shows that Kohlbach index remains constant with signal intensityof about 9 or more.

The results of the above experiments confirm that malt with ESR signalintensity between 9 and 15 is modified enough and satisfies the generalrequirement level of malt quality.

According to the above experimental results, germination state of maltcan be estimated and evaluated based on the ESR signal intensity of thegerminating malt (green malt).

The modification and/or diastatic power of malt can be evaluated bysampling green malt or malt during malting, and measuring ESR signalintensity using the ESR analysis. If the ESR signal intensity is equalto or more than a specific level, the malt quality can be guaranteed.Such a quality evaluation method can be used as a rapid and simplemethod to check the malt quality.

The ESR analysis of malt does not require lyophilizing the malt sample.The malt sample is ground and put in an ESR sample tube. The measurementby the ESR spectrometer takes less than two minutes. Although thesetting of the ESR spectrometer takes about 30 minutes, the ESR analysisrequires substantially less time than conventional method does. Thepreparation of malt samples is quite easy too.

Preferred embodiments of the present invention are described in detailabove. However, the present invention is not limited to theseembodiments, and variations and modifications may be made withoutdeparting from the scope of the present invention.

As is apparent from the above detailed description, the presentinvention makes it possible to evaluate the quality of green malt basedon the ESR signal intensity of green malt sample using electron spinresonance method.

The present invention also makes it possible to evaluate the quality ofmalt based on the ESR signal intensity of malt sample using the electronspin resonance method.

Additionally, the present invention makes it possible to estimateparameters indicating malt modification such as Kohlbach index, Hartongindex at 45° C., diastatic power, β-glucan content, viscosity, andfriability based on parameters measured by the electron spin resonancemethod.

1. A quality evaluation method of green malt by electron spin resonance(ESR) spectrometry, comprising: sampling green malt in a germinationprocess of barley; measuring a peak height of spectrum of the sampledgreen malt at a g value at which an unpaired electron derived from acarbon radical is resonant; determining an ESR signal intensity that isa ratio of the measured peak height of spectrum to a peak height ofspectrum of a reference, per a unit weight of the sampled green malt;and evaluating a germination state of the sampled green malt bycomparing the determined ESR signal intensity with a predeterminedreference level.
 2. A quality evaluation method of malt by electron spinresonance (ESR) spectrometry, comprising: sampling malt in a germinationprocess of barley; measuring a peak height of spectrum of the sampledmalt at a g value at which an unpaired electron derived from a carbonradical is resonant; determining an ESR signal intensity that is a ratioof the measured peak height of spectrum to a peak height of spectrum ofa reference, per a unit weight of the sampled malt; and evaluating amodification state of the sampled malt by comparing the determined ESRsignal intensity with a predetermined reference level.
 3. An evaluationmethod of modification state of malt comprising: determining acorrelation between a parameter measured by general analytical methodsincluding chemical analytical methods, said parameter indicating amodification state of the malt, and an ESR signal intensity of thesampled malt, the ESR signal intensity being determined by an electronspin resonance spectrometry whereby a peak height of spectrum of themalt is measured at a g value at which an unpaired electron of a carbonradical is resonant, and the ESR signal intensity is a ratio of themeasured peak height of spectrum to a peak height of spectrum of areference, per a unit weight of the malt; sampling malt in a germinationprocess of barley; and evaluating said modification of said sampled maltbased on said parameters determined using the corresponding ESR signalintensity determined by said electron spin resonance spectrometry. 4.The evaluation method as claimed in claim 3, wherein said parameter isat least one of a Kohlbach index, Hartong index at 45°C., diastaticpower, β-glucan content, viscosity, and friability.